EP0444092B1 - Preparation de lipase immobilisee sur particules, son procede de production et son utilisation - Google Patents

Preparation de lipase immobilisee sur particules, son procede de production et son utilisation Download PDF

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Publication number
EP0444092B1
EP0444092B1 EP89912889A EP89912889A EP0444092B1 EP 0444092 B1 EP0444092 B1 EP 0444092B1 EP 89912889 A EP89912889 A EP 89912889A EP 89912889 A EP89912889 A EP 89912889A EP 0444092 B1 EP0444092 B1 EP 0444092B1
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EP
European Patent Office
Prior art keywords
lipase
immobilized lipase
immobilized
particulate
diameter
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
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EP89912889A
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German (de)
English (en)
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EP0444092A1 (fr
Inventor
Sven Pedersen
Tomas Tage Hansen
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Novozymes AS
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Novo Nordisk AS
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Priority to AT89912889T priority Critical patent/ATE91503T1/de
Publication of EP0444092A1 publication Critical patent/EP0444092A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • the particulate immobilized lipase according to the invention with macroporous silica or silicates as a carrier material is characterized by the fact that more than 90% of the particles have particle sizes between 100 and 1000 ⁇ m, wherein more than 80% of the pores in the particles exhibit a diameter between 10 and 45 times the diameter of the lipase globules, and wherein the water content of the particulate immobilized lipase is between 1 and 20%, preferably between 2 and 20%, more preferably between 5 and 20%.
  • the invention is exclusively directed to particulate immobilized lipases, and lipases are quite extraordinary enzymes in the sense that the enzymatic activity is functioning on an interface between two phases, meaning that the immobilization of the lipases is a very delicate problem, which highly limits the utility of known immobilization techniques in the field comprising lipase immobilization, vide J. lavayre et al ., Preparation and Properties of Immobilized Lipases, Biotechnology and Bioengineering, vol. XXIV, pp. 1007 - 1013 (1982), John Wiley & Sons.
  • the term "macroporous" means that the pores are at least 250 ⁇ in diameter.
  • the pore diameter is measured by means of the B.E.T. method.
  • the carrier material used in the immobilized lipase according to the invention consists of at least 65% weight of silica and/or silicates, preferably at least 90% by weight of silica and/or silicates.
  • silica or silicates means genuine silica or silicates, i.e. silica or silicates, which are not derivatized.
  • the diameter of the lipase globules can be measured by means of X-ray diffraction analysis and other methods, as indicated in "Biochemistry" of Albert L. Lehninger, 1970, Worth Publishers Inc., page 142 - 143.
  • the diameter of the lipase globules is generally around 50 ⁇ .
  • the particulate silica gels described in the Grace information leaflet SG BC 1E/June 1987 (from Grace, Grace Plaza, 1114 Avenue of the Americas, New York, N.Y. 10036-7794) are well suited for the purpose of the invention, as most of them have pore diameters of 500 ⁇ or above.
  • silica gels can be used for immobilization of cells and enzymes, there is no indication in the leaflet whatsoever that the silica gels can be used for immobilization of lipases, and lipases are exceptional enzymes which in comparison to other enzymes exhibit unique characteristics in regard to immobilization, as explained earlier in this specification.
  • EP patent No. 147,914 is described an immobilized lipase preparation on which lipase is immobilized on a glass carrier with particle size 30-45 mesh and an average pore size of 400 ⁇ .
  • a coupling agent of the organotitanate type is used for the production of the immobilized lipase preparation.
  • the immobilized lipase according to the invention more than 80% of the pores in the particles exhibit a diameter between 12 and 40 times the diameter of the lipase globules. In this interval of pore sizes the expressed lipase activity, measured in BIU/g, is especially high.
  • the lipase is a thermostable lipase.
  • the immobilized lipase can be used in columns operating with high temperature, whereby at least two advantages are obtainable: in the first place it is possible to use the immobilized lipase, e.g. for interesterification, without a solvent, due to the relatively low viscosity of the reaction mixture, in the second place the reaction rate will be relatively high, and in the third place the diffusion rate of substrate and products inside the pores will be increased.
  • the lipase is produced by cultivation of a microorganism containing a gene encoding for and expressing a lipase derived from a strain of Humicola species, Candida antarctica or Rhizomucor miehei . These lipases have been tested, and they all perform well at high temperature in a column.
  • the invention comprises a method for production of a particulate immobilized lipase according to the invention, wherein an aqueous solution of a microbial lipase is contacted with a particulate carrier material, which is macroporous silica or silicates, in which more than 90% of the particles have sizes between 100 and 1000 ⁇ m, and in which more than 80% of the pores in the particles exhibit a diameter between 10 and 45 times the diameter of the lipase globules, during a period of time sufficient to bind the wanted amount of lipase to the carrier material, whereafter the thus formed particulate immobilized lipase is separated from the aqueous phase and the separated immobilized lipase is dried to a water content of between approximately 2 and 20%. It has been found that a wash of the particulate immobilized lipase between the separation thereof from the aqueous phase and the drying thereof is advantageous.
  • a particulate carrier material which is macroporous silica or silicate
  • the period of time which is sufficient to bind the wanted amount of lipase to the carrier material varies from lipase to lipase, and it can range between a few minutes and a few days.
  • the pH during contact between carrier material and aqueous solution does not deviate more than 1 pH unit from the optimal loading pH of the lipase in question in regard to expressed lipase activity.
  • the expressed lipase activity measured in BIU/g of immobilized lipase, is as large as possible.
  • the separation is performed by simple filtration. This is the simplest and cheapest way of performing the separation.
  • the invention comprises a use of the immobilized lipase according to the invention.
  • the use comprises a method for interesterification of fats, wherein liquid fats or fatty mixtures, including free fatty acids or fatty acid esters, are contacted with the immobilized lipase preparation according to the invention, a method for hydrolysis of fats, wherein triglycerides and water are contacted with the immobilized lipase preparation according to the invention, and a method for synthesis of glycerides or other fatty acid esters, wherein a mixture of glycerol or substituted glycerols or other types of alcohols and free fatty acids is contacted with the immobilized lipase preparation according to the invention.
  • no solvent is necessary but a solvent can be used, if desired.
  • the uses may be carried out continuously, e.g. in columns, or batchwise.
  • the lipase activity unit (LU) is determined as described in the publication AF 95.1/2.GB of 83-01-03, obtainable from Novo-Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark.
  • Fig. 1 illustrates the dependency between expressed lipase activity (BIU/g) and pH during loading of a lipase on carrier materials with different pore sizes.
  • Fig. 2 shows the logarithm to the flow rate versus time for a lipase on carrier materials with different pore sizes.
  • the data on Fig. 1 originated as follows.
  • the silica carrier which is a carrier product from Grace described in Biocatalyst Supports SG BC 1E/June 1987 is washed with buffer at the pH to be used during the following lipase adsorption step, i.e. at pH 4, 4.5, 5, 6 and 7, vide Fig. 1, for half an hour and filtered.
  • the wanted amount of lipase which is the lipase activity sufficient for generation of a loading of 186.000 LU/g is dissolved in 5 ml of deionized water and added to 1 g of carrier.
  • the lipase is produced as indicated in Example 1 in Danish patent application no. 4417/86, i.e. by means of Humicola lanuginosa .
  • the pH value is adjusted and the carrier and the lipase solution is slowly agitated by rotation for two hours followed by vacuum filtration.
  • the filtrate is analyzed for hydrolytic activity (LU/ml) in order to determine the amount of adsorbed (loaded) lipase.
  • the immobilized lipase is air dried, the moisture content is adjusted to 10% by weight, and the sample is analyzed for batch interesterification activity (BIU/g). It clarly appears from the figure that the silica carrier with pore size 1500 ⁇ (i.e. 25 times the diameter of the lipase globules), i.e. inside the claimed interval for pore sizes) exhibits an excellent expressed lipase activity at the optimum pH.
  • Fig. 2 On Fig. 2 the three of the four immobilized lipase preparations from Fig. 1 which are inside the scope of the present invention were tested in a column in the following manner.
  • a system consisting of a precolumn containing ion exchange resin saturated with water and an enzyme column (containing 4.5 g immobilized lipase preparation) in series was set up.
  • the function of the precolumn was saturation of the substrate with water.
  • An oil mixture consisting of 28.6% (w/w) lauric acid and 71.4% (w/w) soy bean oil was pumped through the columns.
  • the temperature in the columns was kept at 60°C.
  • the flow rate was adjusted in order to keep a constant conversion of 14% incorporated lauric acid in the soy bean oil.
  • TG is an abbreviation for triglyceride.
  • silica carrier with pore size 1500 ⁇ performs very well in regard to initial flow rate.
  • Immobilized lipase preparations were prepared with Grace 6 as the carrier.
  • the lipase was the Humicola lanuginosa lipase. Except for the drying the immobilized preparations were prepared as indicated in relation to Fig. 1, at pH 4.5.
  • Example 1 are production examples;
  • Example 3, 4, 5, and 6 are application examples, and
  • Example 8 is a comparison example.
  • This example is a comparison example, due to the fact that the pore diameter of the carrier is less than 5 times the diameter of the lipase globules.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Préparation de lipase immobilisée sur particules, dont le matériau porteur macroporeux, qui est à base de silice ou de silicate, se compose de pores ayant un diamètre entre 5 et 45 fois le diamètre de la molécule de lipase. Ladite préparation de lipase, qui est peu coûteuse et présente une activité hautement spécifique, est produite en mettant en contact ledit matériau porteur et une solution aqueuse de lipase. On peut utiliser ladite préparation de lipase pour l'interestérification, l'hydrolyse et la synthèse de matières grasses.

Claims (12)

  1. Lipase microbienne immobilisée sur particules avec un support macroporeux consistant en au moins 65% de silice ou de silicates, dans laquelle plus de 90% des particules possèdent des tailles de particule entre 100 et 1000 µm, dans laquelle plus de 80% des pores dans les particules présentent un diamètre entre 10 et 45 fois le diamètre des globules de lipase et dans laquelle la teneur en eau de la lipase immobilisée sur particules se situe entre 1 et 20%, de préférence entre 2 et 20%, plus préférablement entre 5 et 20%.
  2. Lipase immobilisée sur particules selon la revendication 1, dans laquelle plus de 90% des particules possèdent des tailles entre 200 et 800 µm, de préférence entre 200 et 400 µm.
  3. Lipase immobilisée sur particules selon la revendication 1 ou 2, dans laquelle plus de 80% des pores dans les particules présentent un diamètre entre 12 et 40 fois le diamètre des globules de lipase.
  4. Lipase immobilisée sur particules selon les revendications 1 à 3, dans laquelle la lipase est une lipase thermostable.
  5. Lipase immobilisée sur particules selon les revendications 1 à 4, dans laquelle la lipase est produite par culture d'un micro-organisme contenant un gène codant pour et exprimant une lipase dérivée d'une souche de l'espèce humicola, de candida antarctica ou de rhizomucor miehei.
  6. Procédé pour la production d'une lipase immobilisée sur particules selon les revendications 1 à 5, dans lequel une solution aqueuse d'une lipase microbienne est mise en contact avec un support sous forme de particules qui est de la silice ou des silicates macroporeux, dans lequel plus de 90% des particules possèdent des tailles entre 100 et 1000 µm et plus de 80% des pores dans les particules présentent un diamètre entre 10 et 45 fois le diamètre des globules de lipase, pendant une période de temps suffisante pour fixer la quantité de lipase souhaitée au support, après quoi la lipase immobilisée sur particules ainsi formée est séparée de la phase aqueuse et la lipase immobilisée séparée est séchée jusqu'à une teneur en eau entre 1 et 20%, de préférence entre 2 et 20%, plus préférablement entre 5 et 20%.
  7. Procédé selon la revendication 6, dans lequel la proportion entre la quantité de la solution aqueuse de la lipase microbienne et le poids de support correspond à 10.000 - 500.000 UL/g de support (poids sec).
  8. Procédé selon la revendication 3, dans lequel le pH au cours du contact entre le support et la solution aqueuse ne s'écarte pas de plus de 1 unité de pH du pH de charge optimal de la lipase en question par rapport à l'activité lipase exprimée.
  9. Procédé selon les revendication 6 à 8, dans lequel la séparation s'effectue par simple filtration.
  10. Procédé pour l'interestérification de graisses, dans lequel des graisses ou des mélanges de graisses liquides, y compris des acides gras libres ou des esters d'acides gras, sont mis en contact avec la préparation de lipase immobilisée selon les revendications 1 à 5.
  11. Procédé pour l'hydrolyse de graisses, dans lequel des triglycérides et de l'eau sont mis en contact avec la préparation de lipase immobilisée selon les revendications 1 à 5.
  12. Procédé pour la synthèse de glycérides ou d'autres esters d'acides gras, dans lequel un mélange de glycérol, de glycérols substitués ou d'autres types d'alcools et d'acides gras est mis en contact avec la préparation de lipase immobilisée selon les revendications 1 à 5.
EP89912889A 1988-11-16 1989-11-15 Preparation de lipase immobilisee sur particules, son procede de production et son utilisation Expired - Lifetime EP0444092B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT89912889T ATE91503T1 (de) 1988-11-16 1989-11-15 Auf teilchen immobilisierte lipasezubereitung, verfahren zur herstellung und deren verwendung.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK6386/88 1988-11-16
DK638688A DK638688D0 (da) 1988-11-16 1988-11-16 Partikelformet immobiliseret lipase-praeparat, fremgangsmaade til fremstilling deraf og anvendelse deraf

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EP0444092A1 EP0444092A1 (fr) 1991-09-04
EP0444092B1 true EP0444092B1 (fr) 1993-07-14

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US (1) US5342768A (fr)
EP (1) EP0444092B1 (fr)
JP (1) JP2873251B2 (fr)
CA (1) CA2003163A1 (fr)
DE (1) DE68907611T2 (fr)
DK (1) DK638688D0 (fr)
WO (1) WO1990005778A1 (fr)

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AU1806595A (en) * 1994-02-21 1995-09-04 Novo Nordisk A/S Method for production of an immobilized enzyme preparation and use of the immobilized enzyme preparation
TR199701705T1 (xx) * 1995-06-27 1998-04-21 Unilever N.V. Trigliserid ya�lar�n i�lenmesinde hareketsiz enzim ve kullan�m�.
JP2862509B2 (ja) * 1996-05-28 1999-03-03 東洋電化工業株式会社 リパーゼ固定化用担体及び固定化リパーゼ
SK80799A3 (en) * 1996-12-19 2000-03-13 Unilever Nv Immobilized enzyme and its use for the processing of triglyceride oils
IL129086A0 (en) * 1999-03-22 2000-02-17 Enzymotec Ltd Surfactant-lipase complex immobilized on insoluble matrix
WO1999015689A1 (fr) * 1997-09-24 1999-04-01 Enzymothec Ltd. Complexe surfactant-lipase immobilise sur une matrice insoluble
IL134717A0 (en) * 2000-02-24 2001-04-30 Enzymotec Ltd Method for increasing the performance of immobilized biocatalysts, and catalysts obtained thereby
JP2004344240A (ja) * 2003-05-20 2004-12-09 Takasago Internatl Corp 消臭方法
MY134420A (en) * 2004-02-18 2007-12-31 Univ Putra Malaysia Upm Enantioselective immobilized lipase
JP4220957B2 (ja) * 2004-11-12 2009-02-04 花王株式会社 固定化酵素の製造方法
AU2006256131B2 (en) * 2005-06-09 2010-09-23 Novozymes A/S Lipase powder composition
CA2567576A1 (fr) * 2005-11-10 2007-05-10 Archer-Daniels-Midland Company Methodes de production de monoesters de polyethyleneglycol au moyen d'une lipase
JP4917349B2 (ja) * 2006-05-11 2012-04-18 日清オイリオグループ株式会社 リパーゼ活性の回復方法
ITMI20070435A1 (it) 2007-03-05 2008-09-06 Innovate Biotechnology Srl 2',3'-di-o-acil-5-fluoronucleosidi
US9416383B2 (en) * 2007-04-27 2016-08-16 University Of North Carolina At Chapel Hill Method for enhancing catalytic activity of a lipase
CN104212844A (zh) 2007-06-01 2014-12-17 索拉兹米公司 在微生物中生产油
DE102007027206A1 (de) 2007-06-13 2008-12-18 Süd-Chemie AG Immobilisierung von Enzymen auf Bleicherden
DE102007027195A1 (de) 2007-06-13 2008-12-18 Süd-Chemie AG Thermisch modifizierte Tonmineralien als Trägermaterialien für Enzyme
US7883882B2 (en) 2008-11-28 2011-02-08 Solazyme, Inc. Renewable chemical production from novel fatty acid feedstocks
US20100297749A1 (en) * 2009-04-21 2010-11-25 Sapphire Energy, Inc. Methods and systems for biofuel production
WO2011150410A2 (fr) 2010-05-28 2011-12-01 Solazyme, Inc. Huiles spécifiques produites à partir de microorganismes hétérotrophes recombinants
US9249436B2 (en) 2011-02-02 2016-02-02 Solazyme, Inc. Tailored oils produced from recombinant oleaginous microorganisms
SG11201406711TA (en) 2012-04-18 2014-11-27 Solazyme Inc Tailored oils
JP6168275B2 (ja) * 2012-12-05 2017-07-26 国立研究開発法人産業技術総合研究所 炭酸カルシウム・マイクロカプセル固定化リパーゼ
US9567615B2 (en) 2013-01-29 2017-02-14 Terravia Holdings, Inc. Variant thioesterases and methods of use
US9816079B2 (en) 2013-01-29 2017-11-14 Terravia Holdings, Inc. Variant thioesterases and methods of use
US9783836B2 (en) 2013-03-15 2017-10-10 Terravia Holdings, Inc. Thioesterases and cells for production of tailored oils
US9290749B2 (en) 2013-03-15 2016-03-22 Solazyme, Inc. Thioesterases and cells for production of tailored oils
EP2993993A2 (fr) 2013-04-26 2016-03-16 Solazyme, Inc. Huiles à teneur faible en acides gras polyinsaturés et leurs utilisations
RU2539101C2 (ru) * 2013-05-07 2015-01-10 Федеральное государственное бюджетное учреждение науки Институт катализа им. Г.К. Борескова Сибирского отделения Российской академии наук Биокатализатор, способ его приготовления и способ переэтерификации растительных масел с использованием этого биокатализатора
SG11201602638SA (en) 2013-10-04 2016-05-30 Solazyme Inc Tailored oils
US9765368B2 (en) 2014-07-24 2017-09-19 Terravia Holdings, Inc. Variant thioesterases and methods of use
CN107208103A (zh) 2014-09-18 2017-09-26 泰拉瑞亚控股公司 酰基‑acp硫酯酶及其突变体
JP2018512851A (ja) 2015-04-06 2018-05-24 テラヴィア ホールディングス， インコーポレイテッド Ｌｐａａｔアブレーションを有する油産生微細藻類
US11926739B2 (en) 2021-07-26 2024-03-12 Mcpu Polymer Engineering Llc Modified lignin products for rigid foams
CN119013412A (zh) 2022-04-20 2024-11-22 诺维信公司 用于生产游离脂肪酸的方法

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DK317483D0 (da) * 1983-07-08 1983-07-08 Superfos As Immobiliseret enzympraeparat og fremgangsmade til fremstilling deraf
DK402583D0 (da) * 1983-09-05 1983-09-05 Novo Industri As Fremgangsmade til fremstilling af et immobiliseret lipasepraeparat og anvendelse deraf
ATE117018T1 (de) * 1986-10-17 1995-01-15 Novo Nordisk As Positionsmässig nicht spezifische lipase von candida-arten; verfahren für ihre herstellung und ihre verwendung.

Also Published As

Publication number Publication date
WO1990005778A1 (fr) 1990-05-31
US5342768A (en) 1994-08-30
EP0444092A1 (fr) 1991-09-04
DE68907611T2 (de) 1993-10-21
JPH04501664A (ja) 1992-03-26
JP2873251B2 (ja) 1999-03-24
DE68907611D1 (de) 1993-08-19
CA2003163A1 (fr) 1990-05-16
DK638688D0 (da) 1988-11-16

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